High throughput assays for transmissible spongiform encephalopathies (tse)

ABSTRACT

Stable cell lines which produce the pathological form of PrP after infection with the infectious agent for CJD provide a high throughput assay to identify suitable treatment protocols and compositions. The stable cell lines also provide rich source of infectious CJD agent. They also may be used to identify vaccine candidates. Co-culture of neuronal cells with cells to be tested for infection with a TSE agent also provides a high throughput method for identifying infected cells.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. Ser. No. 11/114,411 filed 25Apr. 2005, which claims priority from U.S. Ser. No. 60/564,748 filed 23Apr. 2004 and from U.S. Ser. No. 60/574,611 filed 25 May 2004. Thecontents of these documents are incorporated herein by reference.

STATEMENT OF RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH

This work was supported in part by grants from the National Institutesof Health and the Department of Defense. The U.S. government has certainrights in this invention.

TECHNICAL FIELD

The present invention relates to compositions and methods foridentifying suitable treatment protocols and treatment compositions fortransmissible spongiform encephalopathies (TSE). More specifically, theinvention relates to stable cell lines which produce the pathologicalform of PrP (PrP-res) after infection with the infectious agent for ahuman or other mammalian form of transmissible spongiformencephalopathy, as well as to such stable cell lines which have furtherbeen modified to become resistant to an antibiotic. The invention alsorelates to the use of the stable cell lines in assays to identifycompositions and/or protocols for treating transmissible spongiformencephalopathy, and to assess the ability of weaker strains of TSE tobehave as vaccines. These cell lines also permit discrimination betweenvarious strains of TSE in a more efficient manner than whole animalassays.

BACKGROUND ART

Creutzfeldt-Jakob disease (CJD) is the human form of a group ofneurodegenerative diseases collectively known as transmissiblespongiform encephalopathies (TSE). These conditions are characterized byhigh levels of the abnormal form prion protein (PrP) i.e., PrP-res, andhave counterparts in bovine spongiform encephalopathy (BSE) and scrapiein sheep. The infectious agents for these conditions have not beenidentified, but it is known that the infection can be transmittedthrough the food supply as well as through peripheral routes includingwhite blood cells in experimental animals and humans. Other animalsbesides humans, sheep and cattle have also been affected by the same oranalogous agents, including pigs, rodents, primates, cats and variouszoo animals. See, for example, Manuelidis, L., Science (1997) 277:94-98.There is no known treatment for these conditions and because humans areat risk with respect to this TSE, the human form is particularly ofinterest. It would be helpful to provide a high throughput assay toevaluate protocols and compositions that would be useful in treating orpreventing this condition in humans and other mammals. The presentinvention employs successful cell lines as models for the various,including human, forms of this disease.

Cell lines have been prepared from neuroblastoma cell lines that areinfected with a scrapie agent which appear to be unstable. Race, R.,Curr. Top. Microbiol. Immunol. (1991) 172:181-193; Borchelt, D., et al.,J. Cell Bio. (1990) 743-752. Further, the characteristics of these cellsas indicative of infection appear to be unreliable, although thesecultures are useful in elucidating metabolism of PrP-res and removal ofPrP-res after treatment with selected compounds. See, Priola, S., etal., Infect. Agents Dis. (1994) 23:54-58; Vey, M., et al., Proc. Natl.Acad. Sci. USA (1996) 93:14945-14949; and Mange, A., et al., J. Virol.(2000) 74:3135-3140. Only infectivity related to scrapie has beenevaluated in these cell lines, however. The murine hypothalamic GT1 andN2a cell lines have proved more stable with respect to scrapie, but onlywith specific strains. Bosque, P., et al., J. Virol. (2000)74:4377-4386.

The present invention resides in creating cell lines stably expressingthe TSE phenotype, including the appearance of PrP-res. These cell linesare useful in assays to identify compounds that will be effective intreating or preventing the development of TSE, e.g., CJD in humans.Previous reports of reproducibly infected murine hypothalamic GT1-7cells (hereafter called GT cells) with a number of different scrapie andCJD agents that had been serially passaged in mice are set forth inNishida, N., et al., J. Virol. (2000) 74:320-325; Arjona, A., et al.,Proc. Natl. Acad. Sci. USA (2004) 101:8768-8773. Stable infection of GTcells did not require cell cloning.

As will be evident below, the invention, in addition to providing celllines stably infected with a TSE agent for evaluating candidatetreatments or for preparing large amounts of the agent itself, alsoconcerns assays to identify potential vaccine candidates as well asmethods for identifying a particular strain of agent based on previouslyobserved behavior. These assays reflect the ability of weaker forms ofan infectious agent to effect a sort of immunity with regard to morevirulent forms, analogous to, for example, vaccination against smallpox,or the use of attenuated virus as a vaccine against polio. Previous workin vivo indicates that similar effects may be seen in TSE infectiveagents. For example, two strains of CJD have been identified—anattenuated SY strain and a more virulent FU strain. Manuelidis, L., etal., Proc. Natl. Acad. Sci. USA (2003) 100:5360-5365. The ability of theSY strain to protect against infection by the FU strain was demonstratedin mice (ibid.).

In TSEs such as human Creutzfeldt-Jakob Disease (CJD), sheep scrapie,and bovine spongiform encephalopathy (BSE), typical cellular andadaptive immune responses to a foreign infectious agent have not beendetected (Manuelidis, L., et al., Science (1997) 277:94-98). Thus thediscovery that SY was able to prevent superinfection by the morevirulent and rapidly lethal FU agent in mice was surprising (Manuelidis,L., Proc. Natl. Acad. Sci. USA (1998) 95:2520-2525). FU and SY are CJDagents isolated from geographically separate regions of the world. Whenpassaged in mice, these agents were readily distinguished by profounddifferences in the incubation time to disease, as well as by thedistribution of brain lesions. The attenuated “slow” SY agent,propagated from a sporadic CJD case, produced only small medial thalamiclesions, whereas the virulent “fast” FU strain caused widespread severelesions.

Even with an intracerebral route of inoculation, there was an obviousprotective effect of SY against high doses of FU challenge agent.Control mice, first inoculated with uninfected brain and then with FU,all developed widespread FU disease >110 days before any clinical signswere seen in SY protected mice (Manuelidis (1998), supra), andrepresentative brains of SY protected mice failed to show even trace FUagent (Manuelidis, L., et al., Neurosci. Lett. (2000) 293:163-166).Moreover, when lower doses of SY doses preceded FU challenge, mice livedwithout any TSE disease for their entire normal lifespan of >600 days.In contrast, parallel mock inoculated mice all died prematurely offulminant FU disease 325 days earlier. Similarly clear protectiveeffects of SY were also seen with peripheral routes of infection(Manuelidis (2003), supra).

No comparable interference has been verified using other TSE strains(Manuelidis (1998), supra). Only a small −35 day increase in incubationtime was observed with the scrapie strains 22L the 22A inoculatedintracerebrally in mice (Dickinson, A., et al., Nature New Biol. (1972)237:244-245). This raised the possibility that the SY CJD agentpossessed a unique ability to protect the host.

It would be helpful to have a convenient assay system to determine thepossibility that weaker strains of TSE agents can exert a protectiveeffect on infection by more virulent ones. The stably infected celllines of the invention provide such a high throughput system.

DISCLOSURE OF THE INVENTION

The invention is directed to modified GT1 sublines and N2a cell linesthat overexpress murine PrP that have been modified to model theprogression of TSE, especially CJD. In one embodiment, the host celllines are those described in Nishida, N., et al., J. Virol. (2000)74:320-325 and include the murine GT1-1 and GT1-7 sublines (GT1sublines), and N2a58 neuroblastoma cells.

The invention also includes an important in vitro assay system to detectand identify TSE infective agents harbored in cells that are infective,but that do not produce PrP-res. These cells, such as blood cells andhomogenates of various tissues, could previously be assessed in animalstudies which require lengthy time periods of up to a year in order toevaluate. The same co-culturing and PrP-res techniques described belowin Example 4 are applied to co-cultures of compositions containing cellssuspected of harboring TSE infective agents with neuronal derived cellsthat will, in response to TSE infection, generate PrP-res. Detection ofinfection can be accomplished within as little as 2.5-3 weeks. In thisassay system, the co-culture is assayed for the presence of PrP-reswhich will result from the transfer of the infective agent from thecomposition to be tested to the cultured neuronal cells. Advantageously,the co-cultured neuronal cells are modified for antibiotic resistance,especially neomycin resistance. This permits assessment of theco-culture after treating with the antibiotic as the compositioncontaining the cells to be tested will be destroyed, while the neuronalco-cultured cells will not. The determination of generation of PrP-resby the neuronal cells, either as to presence or absence or as to level,thus provides results that indicate the presence, absence, or nature ofan infective agent in the co-cultured cellular compositions.

In one aspect the invention is directed to cell lines, such as neuronalcell lines, especially neuroectodermal cell lines, for example GT1-1,GT1-7, and N2a58 cell lines, that are infected with a TSE infectiousagent. The cell lines may also be modified to resist an antibiotic, forexample, by transfecting these cell lines with a plasmid containing anexpression system for an antibiotic resistance gene, such as neomycinresistance.

In another aspect, the invention is directed to a method to identifyagents that are effective in prophylactic or therapeutic treatment ofTSE which comprises contacting a candidate agent with at least one ofthe cell lines of the invention and observing the effect of saidcandidate agent on progress of infection, for example by assessing thelevels of abnormal PrP (PrP-res) present. The level of PrP-res isindicative of the progress of the disease and thus, compounds thatdiminish the level of PrP-res or delay its appearance, as compared tocells treated with control, are identified as agents for treatment ofthis disease. This assay method can be adapted to a high throughputformat, as the times required for disease progression in culture areless than those in vivo and as assessing PrP-res in culture isstraightforward.

In another aspect, the invention is directed to co-cultures of first andsecond cell lines infected stably with different strains of TSE agent.In one embodiment, one of the cell lines is further modified to becomeresistant to an antibiotic that would otherwise be cytotoxic to the hostcell. The co-cultures are useful in evaluating candidate protectiveagents against particular TSE agents. Thus, in one embodiment, one ofthe two cell lines (a target cell line) will contain a candidateprotective TSE agent, and the other a challenge TSE agent. The abilityof the infection by the candidate to protect the host cells againstinfection by the challenge can be assessed by, for example, measuringthe level of PrP-res in the cell line comprising the candidate. Theefficiency of this system can be improved by modifying the candidatecomprising target cell line to be antibiotic resistant so thatsubsequent to challenge, the challenge cell line can be destroyed, andthe effect of the challenge on the target more easily analyzed. Theco-cultures are also useful in distinguishing types of TSE agents basedon previous results of interaction between individual strains. Theinvention is also directed to methods to identify protective agentsusing infected tissue to challenge the stably transfected cellsharboring candidate less virulent TSE agents.

The stably infected cell lines of the invention also provide a richsource of infectious TSE agents, such as CJD agent. Accordingly, anotheraspect of the invention is directed to a method for producing a highnumber of infectious CJD and related TSE agents and a method forproducing and/or isolating a highly concentrated sample of infectiousCJD and related TSE agents. The invention is also directed tocompositions comprising high numbers of infectious CJD and related TSEagents.

In still another aspect, the invention is directed to a method todemonstrate the presence of infectious agents in cells that do notproduce PrP-res. Whole cells, such as white blood cells, can bedemonstrated to be infective by co-culture with target cells thatproduce PrP-res. Previously, the infectivity of these cells, such asblood cells, would be demonstrated on animal experiments where more than300 days are required to obtain results. However, using the co-culturemethod of the invention, the ability to infect co-cultured cells,including co-cultured cells that have been made antibiotic-resistant tosimplify the assay, can be obtained after approximately only threeweeks.

The co-culture methods of the invention have been demonstrated to showinfectivity at dilution levels comparable to those that can be seen inmice; whereas the murine assay takes more than 250 days to see theresults of a 10⁶ dilution of infected brain, the cell culture assayprovides results after 21-28 days at this dilution.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows quantitation of total PrP and the abnormal form thereof(PrP-res) as the cells of the invention are passaged.

FIG. 2 shows Western blots of whole cell lysates from brain and celllines at different passages, showing the pattern obtained as a functionof the passage number and strain.

FIG. 3 shows in situ detection of PrP-res in mock and infected celllines.

FIG. 4 is a diagrammatic representation of the co-culture method of theinvention.

FIG. 5A shows the levels of PrP and PrP-res in target cells co-culturedwith challenge TSE infected cells, where the target cells are wildtype.FIG. 5B shows similar results where the target cells have been infectedwith SY.

FIG. 6A shows results of PrP-res determinations indicating thatinfection of target cells with 22L is protective against FU infection.FIG. 6B demonstrates that infection of target cells with the scrapiestrain Ch is not protective.

MODES OF CARRYING OUT THE INVENTION

The invention provides, for the first time, stably infected cell linesthat can serve as surrogates for animals infected with human or othermammalian forms of TSE. By culturing these cell lines and assessing theeffect of candidate protocols and agents on the progress of infection,typically by measuring the timing and/or amount of their production ofabnormal PrP as compared to controls, successful candidate protocols andcompositions are identified.

The cell lines are obtained by infecting the host cells with infectedbrain homogenates and growing them as described in the examples below.Levels of PrP-res can be detected in situ in the most efficient andrapid form of such assays. Alternatively, the cells may be lysed and thequantity of abnormal PrP in the lysate detected by methods thatdistinguish between normal and abnormal PrP. Details of an illustrativemethod to perform such analyses are set forth in the examples below.

Antibodies specific to the various forms of PrP are known in the art.Abnormal PrP may be recognized by antibodies disclosed by Manuelidis,L., et al., Science (1997) 277:94-98; antibodies which recognize normalPrP amyloid are available for purchase. Alternatively, antibodies,either polyclonal or monoclonal, can be prepared using standardart-known techniques by immunizing suitable subjects and titratingplasma to obtain polyclonal preparations or by immortalizingantibody-producing cells and screening for monoclonal antibodies.

Details of these various techniques are found in the examples below. Itwill be evident that in view of the variability of the appearance ofPrP-res as a function of time, control cells not contacted with thecompositions to be tested should be employed simultaneously with thescreening assay. The assay, however, offers a high throughput method toassess the efficacy of treatment protocols.

The host cells that are infected to obtain the cell lines containingstable infection are preferably derived from the neuronal system, forexample neuroectodermal cells. Illustrated below are two specificcellular types, GT cells and N2a cells; however, the hosts are notlimited to these. Other neuronal cell lines are also suitable hosts. Thestably infected cell lines typically exhibit the phenotype ofTSE-infected cells. Among the features of this phenotype is the presenceof abnormal PrP (or PrP-res). PrP can readily be distinguished fromPrP-res by their differential reactivity with regard to the antibodiesdescribed above, as well as the resistance of abnormal PrP toproteolysis.

A large number of strains of TSE agents are known, which are used hereinto create stable cell lines. Those used in the illustrative samplesbelow include two human strains—a fast-acting strain designated FU, anda slow-acting strain designated SY. Also included in the illustrativeexamples are scrapie agents Ch and 22L. Other agents, such as thoseassociated with BSE and the disease in other mammals can be used aswell. Human TSE strains are of particular interest in view of thecatastrophic health problems caused by the human form of this condition.

One aspect of the invention relates to the ability of weaker strains ofa TSE agent to protect against infection by a more virulent strain. By a“more virulent” strain is meant one that effects higher production ofPrP-res and/or more immediate production of PrP-res and/or results inearlier death or earlier exhibition of other symptoms. For conveniencein the high throughput assays of the invention, the length of timerequired for the appearance of PrP-res and/or the levels of PrP-resproduced are convenient measurements of the progression and virulence ofthe disease. “More virulent” also has an element of host specificity.For example, forms of the agent that are active in sheep, such asscrapie strains, may be less virulent in humans. Thus, the ability of ascrapie strain to protect against infection by a human strain is, ineffect, protection by a less virulent strain.

Candidate compositions and protocols for treatment of a TSE-causeddisease may exert either therapeutic or preventative effects or both.Compositions which might be tested are small molecules, i.e., the typeof molecule generally found among approved drugs in the U.S.pharmacopoeia, as well as peptides, proteins, various nucleic acid-basedcompositions, or any substance or mixture of substances that mightinhibit the progress of the disease. As described below, in one aspect,the candidate composition may be a less virulent form of TSE than thatfor which protection or amelioration is desired. The less virulent formmay be inherently less virulent, or it may derive from the samecausative agent in an attenuated form.

Cultures of stably transformed cells, then, can be characterized ascultures containing target cells where the effect on these target cellsof various perturbations can be assessed. The target cells of theinvention are stably infected with a TSE agent; the candidatecomposition or protocol may be a simple composition or alteration inmedia conditions, temperature, pH, or formulation, that is designed toameliorate the infection. The target cells may also be modified tocontain genes which confer antibiotic resistance. These cells areparticularly useful when assessed in co-culture with cells carryingsubstances or infective agents that are designed to challenge the targetcell. If these challenge cells are not modified for antibioticresistance, the antibiotic may be used to treat the co-culture wherebythe challenge cells are killed, but the target cells are not. Thiseliminates a complicating factor in assessing the progress of infectionin the target cells, as the challenge cells are no longer present tocomplicate the results.

Some of the cell lines stably infected with a TSE agent have beendescribed in Arjona, A., et al., Proc. Natl. Acad. Sci. USA (2004)101:8768-8773. Preferred hosts are neuroectodermal cells.

In a typical experiment, a co-culture of target cells already providedwith a mechanism for resisting infection, for example a weakened form ofthe infective agent, are co-cultured either with challenge cells whichcontain a more virulent form of the agent or with tissue that is alreadyinfected with this more virulent form. In one embodiment, the targetcells are antibiotic resistant, and the challenge is provided byco-cultured infected cells. After co-culture of the target and challengecell lines, the challenge cell line is eliminated by treating theculture with an antibiotic and suitable measurements can then beconducted on the culture with results characteristic of the target cellalone.

As shown below, target cells that contain a weakened form of the humanTSE agent SY exhibit resistance to challenge by a more virulent agent.The evaluation of this protection takes only about three weeks, ascompared to more than 300 days (almost a year) for similar assaysconducted in vivo.

In addition to initial identification of strains of TSE agents that areprotective with respect to more virulent ones, results of these assayscan be used in future determinations to distinguish strains of TSEagents. As shown in the Examples below, the 22L scrapie strain was ableto protect against infection with the more virulent FU strain, whereasthe Ch scrapie strain was not able to do so. Thus, the co-culture assayof the invention can be used to distinguish quickly between these twostrains by assessing their ability to protect against FU.

The residual pure GTneo target cells could then be assayed by Westernblotting for PrP-res, a surrogate marker for positive infection in GTcells. Although PrP-res does not quantitatively correlate withinfectious titers, and may not be detectable in all infectious samples,its presence does indicate infection in GT cells.

This high throughput method for assessment of infection in neuronalcells in culture can also be used to assess the infectivity of cellsthat do not themselves produce PrP-res. Thus, a culture of such cells isco-cultured with cells suspected of containing infection. These cellsmay be blood cells, such as white blood cells, or other types of cellsor tissue homogenates that normally do not produce PrP-res. By theco-culturing method, the neuronal cells, preferably protected bymodification to be antibiotic-resistant, can be assessed for acquiringthe infection from the suspected co-cultured cells after a very shortperiod of about three weeks, as compared to the almost year-long timeframe required for animal experiments. As noted above, the sensitivityof this assay is comparable to that attainable in whole animal assays tolevels of approximately 10⁶ dilutions of infected brain tissue.

As demonstrated in the Examples below, PrP-res is not itself infectious,although abnormal PrP-res is often claimed to be. The Examples show thatcells containing particular TSE agents themselves are infectious andthat certain TSE agents are protective. The protective effect observedcould involve the production of defective interfering particles producedby particular TSE agent strains. Defective viruses have been found inmany viral infections, and they have been associated with both viralpersistence and the modulation of the disease. The attenuated SY agent,that yields very low infectious titers, may generate thousands ofreplication incompetent or empty particles for each complete infectiousparticle. These defective particles could produce a bottleneck atmembrane sites needed for the transit and maturation of the challengeagent. These empty particles would not co-purify with virus-likeinfectious particles, and any strain-specific protein on these particleswould be very difficult to detect among the plethora of complex cellmembrane proteins. Alternatively, the defective particle might be amutant or truncated viral RNA, analogous to the RNAs made byretroviruses that have manifold mechanisms of interference. Even simplerdefective RNAs have been recently described for the small hepatitisdelta satellite virus.

However, applicants do not intend to be bound by any particular theoryto explain the protective effect.

As noted above, the same techniques described in Example 4 in evaluatinginterference with TSE infection by co-culturing target cells withchallenge cells can be used in an assay to detect the presence, absence,nature, or level of TSE infective agent in a cell containing sample. Thecell containing sample is co-cultured with neuronal cells, thuspermitting the transfer of the infective agent from the test cells tothe recipient neuronal cells. As the neuronal cells are able to generatePrP-res, determination of the appearance, non-appearance, or level ofPrP-res can be used as an index to ascertain the infection status of thedonor cells to be tested. This assay system is much more efficient thanthe animal-based systems and permits determination of responses withinthree weeks.

The following examples are offered to illustrate but not to limit theinvention.

Example 1 Preparation of Infected Cell Lines

Murine GT1-1 and GT1-7 sublines from immortalized hypothalamic GT1 cellswere trypsinized and split 1:4 or 1:5 weekly. N2a58 neuroblastoma cellsoverexpressing wt murine PrP were trypsinized and split 1:10 every 5days. These cells are described in Nishida, N., et al., J. Virol. (2000)74:320-325 (supra). Cell lines were fed high glucose DMEM with 10% heatinactivated fetal bovine serum with 1% penicillin-streptomycin (Gibco™,Life Technologies). Cells at 50% confluence in 6-well plates wereexposed for 24 hr to 1% or 2% normal or infected brain homogenates inmedium, washed, grown to 90% confluence, and then transferred to a 25cm² flask (p1). Infected GT1 sublines were not subcloned. N2a58 cellswere diluted at p1 and seeded at 0.7 cells per well in 96-well plates.Single cell clones were screened for PrP-res (detected in 21% of theclones, n=48), and a clone with the strongest signal (FU N2a58H1) wasselected for further expansion in parallel with a clone from mock N2acells (N2a58#1). The amount of total PrP, and of PrP-res, relative tothat found in FU infected mouse brain was determined using whole celllysates to avoid inaccuracies from subcellular fractionation. The leftpanel of FIG. 1 shows the amount of total PrP in brain as a 100%standard per mg cellular protein. The expression of PrP was less in thesublines than in brain, and, as can be seen from the SEM's, varied lessthan 2 fold when sampled every 10-15 passages. Mock controls showed thesame amounts of total PrP (data not shown). The PrP-res in each FUinfected cell line was also less than in brain (right panel), and variedlittle after its first appearance. Notably, PrP-res in differentpassages of GT1-7 and GT1-1 all showed <2 fold changes in PrP-res withprogressive subculture.

Example 2

PrP and PrP-res Patterns in Cell Lines

Whole cell lysates prepared from the cells of Example 1 were treated for30 min with proteinase K (PK) at 25-30 μg/ml to obtain maximal amountsof PrP-res for Western blotting. Protein was determined by Bio-Rad DCprotein assay, and quantitation of PrP bands was done on films in thelinear range. For deglycosylation, SDS denatured proteins were digestedwith 10 units of PNGase F (Sigma) overnight at 37° C. If necessary,proteins of >12 kd were quantitatively precipitated with 6 vol of EtOHand centrifugation at 19,000 g×45 min at 4° C. PrP on blots wasevaluated with four PrP antibodies: i) a rabbit antibody raised againstthe amino terminus (aa23-30) of PrP (IBL Ltd., Japan), ii) a rabbitpolyclonal antibody recognizing PrP-res amyloid (Manuelidis, L., Science(1997) 277:94-98), and iii & iv) two goat polyclonal antibodiesrecognizing the carboxy end of PrP amyloid (aa 91-200; C-20 and M-20,Santa Cruz). Biotinylated lectins (Vector Labs) were used to verifyremoval of sugar residues (Sklaviadis, T., et al., Proc. Natl. Acad.Sci. USA (1986) 83:6146-6150).

Representative data for PrP-res at different passages are shown in FIG.2A. N2a58H cells, with the lowest expression of PrP had the lowestamount of PrP-res, and brain PrP-res was 62%, of total PrP, in goodaccord with previous determinations. Mock controls, treated with normalbrain in parallel, showed no PrP-res (FIG. 2B, lanes 11 & 12, and 2C,lanes 4-6).

Total cellular PrP, as well as PrP-res, had markedly different bandingprofiles by Western blotting in cell lines as compared to brain. In FUand SY infected mouse brain, PrP-res shows identical band mobilities andglycoform ratios and hence does not discriminate these two verydifferent agents. FIG. 2 shows PrP from brain (FIG. 2A, lane 1 & 2B,lane 1) and representative passages of GT1 and N2a sublines (FIG. 2Alanes 2-6 & 2B, lanes 2-4). There are striking differences in intensityand Mr of bands. There are also obvious differences between the Mr ofPrP-res bands as well as their glycoform ratios in brain and cell lines(FIG. 2A, lanes 7-12, 2B, lanes 5-8 & legend). Both FU infected GT1sublines show the same PrP-res pattern, but FU infected GT1-7 has abouttwice the amount of PrP-res as compared to GT1-1 cells (FIG. 2A, lanes8-12). GT1-7 cells challenged with slow SY showed only a weak PrP-ressignal and this appeared only at later passages 13 to 19 (FIG. 2B, lane13). This PrP-res signal was lost after p19 (lane 12). Moreover, the Mrof PrP-res bands from FU and SY in GT1-7 cells were the same in threeindependent analyses, again indicating the PrP-res pattern is cell typerather than agent specific.

To assess if the more resistant amyloid core was retained and all thesusceptible amino-terminal PrP was completely digested, blots wereprobed with antibodies to both the amyloid and amino terminal regions ofPrP. The two amyloid core antibodies showed the same bands whereas afterPK, even at 7× gel loads, no amino terminal PrP was detectable (FIG. 2C,lanes 7-11). The cell type specific PrP-res band patterns did not changewith passage (FIG. 2A). We also tested if the different sizes of PrP-resbands in cells were due to differential glycosylation. Thispost-translational modification has been proposed to encodestrain-specific properties, despite the fact that PrP deglycosylationalters neither the infectious titer nor the strain characteristics of aCJD agent. FIG. 2C, lanes 12-14, shows the complete deglycosylation ofrepresentative PrP-res samples. The higher Mr bands in GT1 cells ascompared to brain were due to increased glycosylation. There was only asingle low Mr band after deglycosylation with PNGase. Lectin stainingalso verified the deglycosylation of PrP-res, but not of many other PKresistant proteins. Despite agent strain differences, deglycosylatedPrP-res was the same in FU and SY infected GT1-7 cells, as in brain.However, GT1 cells showed a 1-2 kd lower Mr than infected brain,presumably caused by sugar residues on PrP during PK digestions. Henceit might be predicted, according to the prion hypothesis, that the agentstrain passaged in GT1 cells should give rise to a variant agent strainwhen inoculated into mice.

Example 3 In situ Detection of Pathologic PrP

Because cells detached and became morphologically disrupted using GdnHCland PK treatments, methods for PrP-res detection in paraffin sectionswere modified. Cells in flasks were fixed in situ with 4% freshparaformaldehyde in PBS for 10 min, scraped, pelleted at 4,000 g×10 min,and then fixed for an additional 12-16 hrs before paraffin embedding.Slides were autoclaved in citrate buffer, digested with 0.002-0.004%trypsin 7-15 minutes, quenched, exposed to antibodies, and developedwith Vector Red. FU and mock infected control cells were mounted on thesame slide.

To visualize PrP-res in individual cells, and thereby estimate thepercentage of infected cells, we evaluated cell pellet sections treatedwith trypsin. Limited trypsin digestion left only the amyloid core ofpathologic PrP intact. FIG. 3 shows representative mock and FU infectedcells probed with an antibody to the carboxy portion of PrP-res. Theamino terminal antibody, as in Western blots of FIG. 2C, showed nosignal, indicating complete removal of non-amyloid portions of PrP (datanot shown). Mock cells displayed no abnormal PrP-res aggregates (toppanel). In contrast, FU infected GT1-7 and GT1-1 cells displayedabundant PrP-res aggregates in >30% of cells. In GT1-7 cells, PrP-resformed compact aggregates within the cytoplasm (arrow, middle panel).About 30% of the cells showed these PrP-res aggregates. This is probablyan underestimate of infected cells since sections will not sample allsuch aggregates. FU infected GT1-1 cells exhibited a strong, but morediffuse staining throughout the cytoplasm (arrows, bottom panel),although a few cells showed the more compact PrP-res aggregates (opentriangle). A high proportion of FU GT1-1 cells were PrP-res positive(≧50%), and since cells were fixed in situ before scraping, the closelyassociated positive cells may reflect cell to cell spread of agent.There were a few more pyknotic nuclei in infected GT1 sublines ascompared to mock controls, possibly due to high levels of infectionand/or secondary to pathologic PrP-res accumulation, but the vastmajority of cells were morphologically normal. Thus these cells can beadvantageous for agent-specific studies, as they are not visiblycompromised by the degeneration found in end-stage brain. Although notquantitative, these in-situ results are consistent with previouslydetermined ratios of 100,000 PrP-res molecules to each infectious dose.

Example 4 Co-Cultures and Protective Effect of Weakened Strains

This example describes the ability of weaker strains of TSE agents tointerfere with infection by more virulent strains at the cellular level,using the stable cell lines of the invention.

Table 1 shows the cell lines used to test interference. The infectiousencephalopathy agents and their source are shown, as well as the numberof cell passages post infection at the time interference was tested. Chis the UK Chandler scrapie agent (often referred to as RML in the USA).FU (also known as FK-1) was isolated from a patient in Japan withGerstmann-Straussler-Sheinker Disease (GSS) and a 102L mutation in hisPrP gene. This FU agent produces widespread brain lesions 120 days aftermouse inoculation ic, while SY produces circumscribed thalamic lesionsonly after 350 days. The Ch and 22L scrapie strains, as FU, also producerapid disease in mice with widespread brain pathology, makinginterference between these strains impossible to assess in vivo.Infectious titers of SY and FU after extended in vitro splits werepreviously determined and FU showed >1,000 fold more infectivity than SYin vitro. Arjona, A., et al., Proc. Natl. Acad. Sci. USA (2004)101:8768-8773 (supra). Bioassay titers for 22 L in cells culturedfor >100 passages was higher than in brain or FU infected cells (resultsreproduced independently in US and Japan, data not shown).

TABLE 1 Cell lines and TSE agents passages Infectious TSE agent postCell line strain origin infection Challenge: GT (GT1-7) — SY + GT SYsporadic CJD in USA ≧106 FU + GT FU GSS 102L in Japan ≧110 Ch + GT Ch(RML) drowsy scrapie in UK ≧50 22L + GT 22L scrapie in UK ≧50 (SSBP/1)Targets: GTneo mock SY + GTneo SY sporadic CJD in USA >110 Ch + GTneo Ch(RML) drowsy scrapie in UK >15 22L + 22L scrapie in UK >15 GTneo(SSBP/1)

The results obtained in the experiments described below can besummarized in Table 2. In this table, +indicates strong PrP-res signal(positive infection), −indicates no detectable PrP-res, and (+/−)indicates ⅓ experiments where there was a very weak PrP-res signal. C-13is a 13 kd band that is diagnostic for FU superinfection.

TABLE 2 Summary of Interference Experiments agent PrP-res targetchallenge PrP-res (total) C-13 Interference Mock FU + + No Mock 22L + −No Mock Ch + − No SY Mock − − No SY FU −(+/−) − Yes SY 22L −(+/−) − YesSY Ch −(+/−) − Yes 22L FU + − Yes Ch FU + + No

As expected, unaltered GT cells were readily infected by FU, 22L, andCH. Target cells that had been modified by the weak TSE agent SY appearprotected against infection by all three tested strains. Small amountsof PrP-res were sometimes seen as described below, but in general,PrP-res was absent. The diagnostic C-13 band in protected targets wasnot present when co-culture with an FU bearing strain was performed.Thus, SY was able to interfere in all cases where challenge wassupplied. Of course, there was nothing to protect against when mockchallenge was done.

Finally, the table shows that 22L was able to protect against FUinfection, but Ch was not.

FIG. 4 outlines in more detail the in vitro interference strategy usedin this example. Target GTneo cells were co-cultured with uninfected(mock) GT, or infected GT challenge cells for two days, and the GTchallenge cells were then killed by G418 antibiotic treatment. (Incontrol experiments GT cells were completely eradicated by G418 in 10days.) At 21 days (5 passages), the screening target GTneo cells werecollected for PrP and PrP-res Western Blot analyses. Assays were alsodone at more extended passages to test if PrP-res increased. Theinfected challenge GT cells used were persistently infected for 1-2years and continued to have high levels of infectivity when assayed bymouse titration. A neomycin resistant plasmid was introduced intopersistently infected SY GT cells for experiments in column 2, and GTneocells were newly infected with scrapie 22L and Ch brain homogenates thenpassaged >15 times before testing interference against FU as outlined incolumn 3.

Neomycin (neo) resistant GT cells, both uninfected and SY-infected, wereestablished by introducing the plasmid pEGFP-C1 (Clonetech) withEffectene transfection reagent (QIAGEN) according to manufacturer'sinstructions. The GT+SY cells had been continuously subcultured morethan 106 passages and shown to be persistently infected. Resistantclonal cells were isolated by selection with 500 μg/ml of G418 (GIBCOBRL). Neo-resistant GT cells were incubated with 0.2% mouse brainhomogenates to produce mock and infected GTneo cells as previouslydescribed (Nishida, N., et al., J. Virol. (2000) 74:320-325) from endstage brain infected with the 22L scrapie agent or the Chandler (RML)scrapie agent. These cells were used for experiments after stableinfection was confirmed by Western blotting for PrP-res at passages 10and 15 in vitro. All the different GT cell lines (GT and GTneo,uninfected and infected) were morphologically the same and showed thesame doubling time. They were passaged as previously described (supra).

GTneo target cells were co-cultured with GT challenge cells that weremock-infected, or infected with the TSE agents shown in Table 1. GT+FUcells have been maintained for more than 2 years and showed persistenthigh titers of infectivity. On the day before starting co-cultureexperiments, 1×10⁶ neo-resistant target cells were plated in a T25flask, and then overlayered with same number of the challenge cells onthe next day. The co-cultures were incubated for 2 days at 37° C., 5%CO₂, in medium without antibiotic. To eradicate challenge GT cells fromthe culture, the cultures were repeatedly split at a 1:3 dilution whilebeing treated with 500 μg/ml G418 at each passage. Some neo-sensitivecells started dying on the first day, but the killing effect of G418 wasgradual, and 60% of GT the cells were eliminated by 5 days of treatment.Therefore significant numbers of challenge cells were present inco-cultures for 7 (2+5) days. We confirmed that neo-sensitive cells werecompletely eradicated by day 10th day of treatment with G418 (byparallel culture). Thus assay for more than 5 passages (3 weeks) in G418antibiotic ensured all PrP-res originated only from GTneo target cells.

After 5 passages the confluent GTneo cells in a T25 flask were washedtwice with cold PBS- and lysed with 500 μl of Cell-lysis buffer: 0.5%Triton X-100, 0.5% sodium deoxycholic acid, 150 mM NaCl, 50 mM TrisHClpH7.5, 2 mM EDTA. Cell lysates were spun at 3,000 RPM for 5 min toremove nuclei and insoluble material. This also removed some large PrPaggregates as determined by Western blotting (data not shown). Afternormalizing the supernatant protein concentration to 1 mg/ml, 500 μl ofeach sample was treated with 10 μg of proteinase K (20 μg/mg protein)(Bohringer) for 30 min at 37° C., and the digestion stopped by adding 3mM PMSF. All the PK-digested samples were then centrifuged at 20,000×gfor 45 min at 4° C., and the pellets were resuspended in 30 μl of1×Laemmli sample buffer. SDS-PAGE and Western blotting were done with 50μg of protein loaded per sample. For detection of PrP in undigestedtotal cell lysates the membrane was incubated with the SAF32 monoclonalantibody that recognizes the octapeptide repeat region (supplied byJaques Grassi, France), and the polyclonal M-20 antibody (Santa CruzBiotech, Calif.) that recognizes a C-terminal region of PrP was used todetect PrP-res. The extra 13 kd C terminal band of PrP-res in FU had notbeen appreciable in digested whole cell lysates previously and thedouble centrifugation (fractionation steps here, as well as the use ofTriton-X 100 rather than NP-40), led to the relative enrichment of thisPrP-res band. It did not bring out the 13 kd band with any of the otherstrains.

Infectivity assays of cells infected with SY and FU were done asdescribed and reported (Arjona (2004), supra), and the same bioassaymethods were used to test 22L infected cells. These cells also hadsimilarly high or higher levels of infectivity per cell than brain andGT+FU cells (independent assays in Japan and USA, data not shown).

FIG. 5 shows the results of co-culture of mock (5A) and SY infected (5B)GTneo cells, as well as with equal numbers of uninfected GT cells (−) orinfected GT+22L, GT+Ch and GT+FU cells as indicated. After 2 daysco-cultures were treated with G148 to select for GTneo target cells andpassaged 5 times before assays for undigested PrP (left panels) anddigested PrP-res (right panels). 1° indicates the primary infection and2°, the challenge infection. PrP-res indicates positive infection. TheC-13 band appeared only in FU infected cells. PK indicates limitedproteinase K digestion, and SAF-32 and M-20 antibodies (Ab) were usedfor PrP and PrP-res detection, respectively. There is minimal or noPrP-res in SY protected cells as compared to controls. Markers in kd areindicated at left.

Thus, FIG. 5A shows a representative example of the large amount ofPrP-res produced by GTneo mock cells after they had been challenged withinfected GT cells and then treated with G418. The left half of thefigure shows undigested PrP in GTneo cells challenged with uninfectedbrain (−), or with GT+22L, GT+Ch, or GT+FU infected cells. All cellsexpress similar amounts of PrP. The right side of the blot shows PrP-resin these detergent lysates after limited proteinase K (PQ digestion andcentrifugation of aggregates at 20,000 g. There is a large amount ofPrP-res produced by cells exposed to each of the infected GT cell lines,whereas no PrP-res is seen in the cells (−) challenged only byuninfected cells. Further in vitro passages of these selected GTneocells did not alter these findings, indicating that the mock cells werepersistently infected by each of the challenge agents. The three majorPrP-res bands are the same in all infected cells. However, FU infectedsamples showed an extra minor 13 kd band (labeled C-13), and thisallowed diagnosis of FU superinfection in subsequent challengeinfection.

In summary, control experiments showed rapid infection with de novoproduction of large amounts of PrP-res by 21 days after co-culturechallenge. (In mice infected with the virulent FU agent intracerebrally,it takes >90 days to detect any PrP-res in brain.) More than 95% ofhomogenate infectivity is cleared from brain within 24 hours and livingcell-to-cell contacts may enhance the transfer TSE agents.

FIG. 5B shows a representative blot of PrP and PrP-res after challengeto SY protected GTneo cells, with protein loads and conditions the sameas in 5A. SY infected cells had no detectable PrP-res, and this greatlysimplified the interference assay, since after challenge by co-culture,a continued lack of PrP-res would indicate this covert SY infectioninterfered with transmissions from GT cells loaded with much higherlevels of challenge agent. It is obvious that there is no detectablePrPres in SY infected cells after co-culture with 22L scrapie infectedcells. The lack of PrP-res also shows that the G418 selection regime wasable to completely remove PrP-res positive challenge cells. The failureto display PrP-res after challenge is strong evidence that the SY agentprevented superinfection by this particular 22L scrapie agent. WithGT+Ch scrapie agent and GT+FU agent challenges, there was a very smallamount PrP-res as compared to mock controls. Such a low level of PrP-resshowed SY infection had substantially interfered with superinfection bythe Ch scrapie and FU agents, but that the protection might not beabsolute. This apparent “leakiness” could be due to the relatively lowinfectivity of SY. Alternatively, the low levels of PrP-res detectedafter Ch and FU challenge could reflect increased activation of the SYagent resulting from the stress of challenge itself. Such increased SYactivation, with consequent host production of pathologic PrP-res cannotbe excluded, because we have found certain chemical stresses alone caneduce similar low levels of PrP-res in persistently infected SY cells(unpublished data).

Further passages did not increase the low amount of PrP-res in eitherthe PrP-res negative cells (−) or in the cells with very low PrP-res(+/−) as summarized in Table 2 alone. Additionally, the 13 kd PrP-resband elicited by FU infection was not detectable in the FU challenged SYcells, and the lack of any distinguishing strain-specific PrP-respatterns for the other three common major PrP-res bands made itimpossible to determine if the low amounts of new PrP-res in were due tolow levels of superinfection, or to increased SY activation. In twoadditional repeat experiments, SY again interfered either completely, oralmost completely, with superinfection by 22L, Ch and FU agents.However, perfect interference was inconsistent with respect to eachagent strain. In the repeat experiments, complete prevention of Ch andFU superinfection was found at least once, and extensive but incompleteinterference was observed once with 22L scrapie agent challenge. Theresults summarized in Table 2 are consistent with animal experimentsshowing dose dependence and/or slight leakiness of the SY infection inthe target cells.

FIG. 6 shows the results of FU challenge of 22L infected GTneo cells(6A) and Ch (6B) scrapie infected GTneo cells. GTneo cells afterchallenge with mock infected cells (−) show equivalent high levels ofPrP-res already present in these cells. After challenge of 22L infectedcells with FU infected GT cells, the pattern and amount of PrP-res isunchanged, indicating no appreciable superinfection. No 13 kd banddiagnostic of FU is detectable. In contrast massive superinfection by FUis obvious in Ch infected cells. The PrP-res is markedly increased, andthe 13 kd band is strong (C-13). The marker for 20 kd is indicated.

In more detail, for the experiments new persistent infections of GTneotarget cells were established by standard application of infected Ch and22L scrapie shown in FIG. 6, brain homogenates, and it was verified thatthese GTneo cells continued to produce substantial amounts of pathologicPrP-res for >15 in vitro passages before challenging them with GT+FUcells. The 22L and Ch scrapie infected GTneo target cells were thenchallenged with FU+GT cells which exhibit the 13 kd band.

The results in FIGS. 6A and 6B show scrapie strains 22L and Ch haddifferent capacities for interfering with FU superinfection. The sameantibiotic selection protocol was used before performing PrP-res assays.FIG. 6A (PrP-res profiles for representative mock and FU challenges totarget 22L+GTneo cells) show PrP-res intensity and band pattern in thetarget 22L cells exposed to FU (22L(FU) were indistinguishable from the22L cells that were not challenged. The lack of the FU linked 13 kd bandfurther confirms that the PrP-res reflected only the resident 22Linfection. The 13 kd PrP-res band also did not appear with further invitro passages, indicating it was not covertly contaminating the targetcells. The protection afforded by 22L scrapie against FU-C,JD agentsuperinfection was complete and reproducible, as no extra PrP-res wasdetectable in two additional repeat experiments.

In sharp contrast, as shown in FIG. 6B, Ch scrapie infection did notprotect GTneo cells from FU superinfection. PrP-res accumulation inthese FU challenged cells (Ch(FU) was considerably more intense than inCh-GTneo cells that were not challenged, and consistent with the amountof pathologic protein that would be provoked by infection with bothagents. The 13 kd band diagnostic of positive FU superinfection was alsopresent. These Ch(FU) results were replicated two additional times.

These data also prove that large amounts of PrP-res itself have noinhibitory effect on superinfection, and that two different TSE agentscan replicate and maintain their identity in a monotypic culture.

Example 5 Effect of Direct Cell Contact in Co-Culture

Because co-culture infection was so rapid and reproducible, additionalexperiments were done to determine if agent transmission was facilitatedby cell-to-cell contact, or was more efficient with cell-freeextracellular particles. Direct cell-to-cell contacts for efficientagent transmission can be important in vivo, as in the transfer ofinfectious agent from follicular dendritic cells to transiting whiteblood cells.

Mouse bioassays of concentrated supernatants collected from both 22L andFU infected GT cells were 1,000 fold less infectious than the remainingwhole washed cells (data not shown). In addition, direct cell-to-cellcontact was prevented by using 0.4u filters between donor and targetcells to permit the transit of large viruses and aggregates, but notwhole cells. As in the co-culture experiments above, equal numbers ofhealthy donor and target cells were planted, and exposure was allowed toprogress for weeks rather than days. When cell-cell contact wasprevented, target cells required more in vitro passages to achieveproduction of PrP-res and target cells continually exposed to donorcells did not always become positive (data not shown), as would bepredicted from the results above.

1. A method to identify an agent potentially effective to treat TSE, themethod comprising: (a) contacting with a candidate agent a co-culturecomprising a mammalian neuronal cell line stably infected with amammalian transmissible spongiform encephalopathy (TSE) agent andtargeted living non-infected neuronal cells modified to be resistant toan antibiotic, wherein said stably infected cell line is infective toneuronal cells in co-culture therewith, and (b) measuring the effect ofthe candidate agent on the level of infection in the targeted neuronalcells, whereby an agent that decreases the level of infection in saidtargeted neuronal cells is identified as an agent potentially effectiveto treat TSE.
 2. The method of claim 1, wherein abnormal prion protein(PrP-res) is present in said cell line, and the concentration of PrP-resin said cell line shows less than 2 fold change with progressivesubculture over at least 15 passages.
 3. The method of claim 1, whereinsaid level of infection is measured as the level of abnormal PrP(PrP-res) in said targeted neuronal cells, and wherein a decrease in thelevel of PrP-res in response to contact with the candidate agentindicates a decrease in the level of infection.
 4. The method of claim1, wherein the co-culture is treated with an antibiotic prior to saidmeasuring the level of infection.
 5. The method of claim 4, wherein saidantibiotic is neomycin.
 6. The method of claim 4, wherein the method isperformed as a high throughput assay.
 7. A method to determine thepresence or absence of cells containing an infective TSE agent in acomposition of which method comprises co-culturing said composition witha neuronal cell line receptive to TSE infection and determining thepresence, absence, or level of PrP-res in said co-cultured neuronal cellline, whereby the presence of PrP-res in the neuronal cell lineindicates the presence of a TSE infective agent containing cells in thecomposition tested.
 8. The method of claim 7, wherein the neuronal cellline has been modified to be resistant to an antibiotic.
 9. The methodof claim 8, wherein the antibiotic is neomycin.
 10. The method of claim8, wherein said co-culture is treated with antibiotic prior todetermining the presence, absence, or level of PrP-res.
 11. The methodof claim 7, wherein the composition to be tested is a homogenate oftissue or comprises isolated blood cells.
 12. The method of claim 7,wherein said determining of the presence, absence, or level of PrP-resis performed within about three weeks from the start of co-culture.